In this project we propose to study the molecular events in the regulation of hemoglobin biosynthesis. Initial studies in Friend mouse erythroleukemia cells demonstrated that the addition of hemin to the tissue culture medium can increase globin chain synthesis and increase globin mRNA content. The erythrodifferentiation in these cells can be blocked by the addition to the culture medium of aminotriazole, an inhibitor of heme biosynthesis. This inhibition can be prevented by the simultaneous addition of hemin to the medium. Current work focuses on the effect of hemin on in vitro translation using extracts of Friend cells. An active translational system was prepared and two fold stimulation of overall incorporation by 100 micrograms/ml hemin was reproducibly demonstrated. When extracts were treated with micrococcal nuclease to obtain an mRNA dependent translational system, 10 to 20 fold stimulation by hemin in the translation of added globin mRNA was obtained. No difference in the effect of hemin on extracts of control or induced cells could be discerned. New studies include efforts to obtain an active system of transcription in nuclei prepared from Friend cells. It is hoped that adequate RNA can be synthesized to quantitate globin mRNA synthesis in vitro in nuclei. The effects of various compounds such as hemin will be studied. Efforts will also be made to obtain a human cell line which undergoes erythrodifferentiation in tissue culture. BIBLIOGRAPHIC REFERENCES: Dabney BJ and Beaudet AL: Increase in globin chains and globin mRNA in erythroleukemia cells in response to hemin. Arch Biochem Biophys 179: 106-112, 1977.